A DNA-PK phosphorylation site on MET regulates its signaling interface with the DNA damage response.

The DNA damage response (DDR) is intertwined with signaling pathways downstream of oncogenic receptor tyrosine kinases (RTKs). To drive research into the application of targeted therapies as radiosensitizers, a better understanding of this molecular crosstalk is necessary. We present here the characterization of a previously unreported MET RTK phosphosite, Serine 1016 (S1016) that represents a potential DDR-MET interface. MET S1016 phosphorylation increases in response to irradiation and is mainly targeted by DNA-dependent protein kinase (DNA-PK). Phosphoproteomics unveils an impact of the S1016A substitution on the overall long-term cell cycle regulation following DNA damage. Accordingly, the abrogation of this phosphosite strongly perturbs the phosphorylation of proteins involved in the cell cycle and formation of the mitotic spindle, enabling cells to bypass a G2 arrest upon irradiation and leading to the entry into mitosis despite compromised genome integrity. This results in the formation of abnormal mitotic spindles and a lower proliferation rate. Altogether, the current data uncover a novel signaling mechanism through which the DDR uses a growth factor receptor system for regulating and maintaining genome stability.

Restriction of extracellular lipids renders pancreatic cancer dependent on autophagy.

BACKGROUND: KRAS is the predominant oncogene mutated in pancreatic ductal adenocarcinoma (PDAC), the fourth cause of cancer-related deaths worldwide. Mutant KRAS-driven tumors are metabolically programmed to support their growth and survival, which can be used to identify metabolic vulnerabilities. In the present study, we aimed to understand the role of extracellularly derived fatty acids in KRAS-driven pancreatic cancer. METHODS: To assess the dependence of PDAC cells on extracellular fatty acids we employed delipidated serum or RNAi-mediated suppression of ACSL3 (to inhibit the activation and cellular retention of extracellular fatty acids) followed by cell proliferation assays, qPCR, apoptosis assays, immunoblots and fluorescence microscopy experiments. To assess autophagy in vivo, we employed the KrasG12D/+;p53flox/flox;Pdx1-CreERT2 (KPC) mice crossed with Acsl3 knockout mice, and to assess the efficacy of the combination therapy of ACSL3 and autophagy inhibition we used xenografted human cancer cell-derived tumors in immunocompromised mice. RESULTS: Here we show that depletion of extracellularly derived lipids either by serum lipid restriction or suppression of ACSL3, triggers autophagy, a process that protects PDAC cells from the reduction of bioenergetic intermediates. Combined extracellular lipid deprivation and autophagy inhibition exhibits anti-proliferative and pro-apoptotic effects against PDAC cell lines in vitro and promotes suppression of xenografted human pancreatic cancer cell-derived tumors in mice. Therefore, we propose lipid deprivation and autophagy blockade as a potential co-targeting strategy for PDAC treatment. CONCLUSIONS: Our work unravels a central role of extracellular lipid supply in ensuring fatty acid provision in cancer cells, unmasking a previously unappreciated metabolic vulnerability of PDAC cells.

ACSL3-PAI-1 signaling axis mediates tumor-stroma cross-talk promoting pancreatic cancer progression.

Pancreatic ductal adenocarcinoma (PDAC) is characterized by marked fibrosis and low immunogenicity, features that are linked to treatment resistance and poor clinical outcomes. Therefore, understanding how PDAC regulates the desmoplastic and immune stromal components is of great clinical importance. We found that acyl-CoA synthetase long-chain 3 (ACSL3) is up-regulated in PDAC and correlates with increased fibrosis. Our in vivo results show that Acsl3 knockout hinders PDAC progression, markedly reduces tumor fibrosis and tumor-infiltrating immunosuppressive cells, and increases cytotoxic T cell infiltration. This effect is, at least in part, due to decreased plasminogen activator inhibitor-1 (PAI-1) secretion from tumor cells. Accordingly, PAI-1 expression in PDAC positively correlates with markers of fibrosis and immunosuppression and predicts poor patient survival. We found that PAI-1 pharmacological inhibition strongly enhances chemo- and immunotherapeutic response against PDAC, increasing survival of mice. Thus, our results unveil ACSL3-PAI-1 signaling as a requirement for PDAC progression with druggable attributes.

PLCγ1 suppression promotes the adaptation of KRAS-mutant lung adenocarcinomas to hypoxia.

Mutant KRAS modulates the metabolic plasticity of cancer cells to confer a growth advantage during hypoxia, but the molecular underpinnings are largely unknown. Using a lipidomic screen, we found that PLCγ1 is suppressed during hypoxia in KRAS-mutant human lung adenocarcinoma cancer cell lines. Suppression of PLCγ1 in hypoxia promotes a less oxidative cancer cell metabolism state, reduces the formation of mitochondrial reactive oxygen species and switches tumour bioenergetics towards glycolysis by impairing Ca2+ entry into the mitochondria. This event prevents lipid peroxidation, antagonizes apoptosis and increases cancer cell proliferation. Accordingly, loss of function of Plcg1 in a mouse model of KrasG12D-driven lung adenocarcinoma increased the expression of glycolytic genes, boosted tumour growth and reduced survival. In patients with KRAS-mutant lung adenocarcinomas, low PLCγ1 expression correlates with increased expression of hypoxia markers and predicts poor patient survival. Thus, our work reveals a mechanism of cancer cell adaptation to hypoxia with potential therapeutic value.

Pharmacological modulation of mitochondrial calcium uniporter controls lung inflammation in cystic fibrosis.

Mitochondria physically associate with the endoplasmic reticulum to coordinate interorganelle calcium transfer and regulate fundamental cellular processes, including inflammation. Deregulated endoplasmic reticulum-mitochondria cross-talk can occur in cystic fibrosis, contributing to hyperinflammation and disease progression. We demonstrate that Pseudomonas aeruginosa infection increases endoplasmic reticulum-mitochondria associations in cystic fibrosis bronchial cells by stabilizing VAPB-PTPIP51 (vesicle-associated membrane protein-associated protein B-protein tyrosine phosphatase interacting protein 51) tethers, affecting autophagy. Impaired autophagy induced mitochondrial unfolding protein response and NLRP3 inflammasome activation, contributing to hyperinflammation. The mechanism by which VAPB-PTPIP51 tethers regulate autophagy in cystic fibrosis involves calcium transfer via mitochondrial calcium uniporter. Mitochondrial calcium uniporter inhibition rectified autophagy and alleviated the inflammatory response in vitro and in vivo, resulting in a valid therapeutic strategy for cystic fibrosis pulmonary disease.

The ACSL3-LPIAT1 signaling drives prostaglandin synthesis in non-small cell lung cancer.

Enhanced prostaglandin production promotes the development and progression of cancer. Prostaglandins are generated from arachidonic acid (AA) by the action of cyclooxygenase (COX) isoenzymes. However, how cancer cells are able to maintain an elevated supply of AA for prostaglandin production remains unclear. Here, by using lung cancer cell lines and clinically relevant KrasG12D-driven mouse models, we show that the long-chain acyl-CoA synthetase (ACSL3) channels AA into phosphatidylinositols to provide the lysophosphatidylinositol-acyltransferase 1 (LPIAT1) with a pool of AA to sustain high prostaglandin synthesis. LPIAT1 knockdown suppresses proliferation and anchorage-independent growth of lung cancer cell lines, and hinders in vivo tumorigenesis. In primary human lung tumors, the expression of LPIAT1 is elevated compared with healthy tissue, and predicts poor patient survival. This study uncovers the ACSL3-LPIAT1 axis as a requirement for the sustained prostaglandin synthesis in lung cancer with potential therapeutic value.

Delay of ejaculation induced by SB-277011, a selective dopamine D3 receptor antagonist, in the rat.

INTRODUCTION: Dopamine (DA) plays a key role in different aspects of the male sexual response, including sexual motivation and arousal, penile erection, and ejaculation. The modalities of action of DA are however unclear, although the various DA receptors may differentially mediate the activity of DA in different aspects of the male sexual response. AIM: To clarify the role of DA D(3) receptors in the control of the male sexual response. METHODS: The effects of a highly selective DA D(3) receptors antagonist (SB-277011; intraperitoneal) were tested in experimental paradigms exploring several aspects of the male sexual response in (i) anesthetized rats using 7-hydroxy-N,N-di-n-propylaminotetralin to induce ejaculation and (ii) conscious rats using sexual incentive motivation and mating tests. MAIN OUTCOME MEASURES: Physiological markers of erection and emission and expulsion phases of ejaculation were measured in anesthetized rats. Behavioral parameters of sexual incentive motivation and mating tests were quantified. RESULTS: In anesthetized rats, we found that SB-277011 specifically and dose-dependently inhibited the expulsion phase of ejaculation without impairing either emission phase or erection, and this resulted in delayed ejaculation. Administration of SB-277011 had no effect on the spontaneous preference that males displayed for sexually receptive females as shown in sexual incentive motivation test. Delayed ejaculation was confirmed when male rats were administered with the highest dose of SB-277011 (10 mg/kg) in mating test, where males were free to copulate with estrous females. In addition, the refractory period following ejaculation was lengthened in rats treated with SB-277011. CONCLUSION: As a whole, the present data demonstrate the specific and primary role of D(3) receptors in the expulsion phase of ejaculation and provide preclinical evidence for the investigation of the therapeutic potential of D(3) antagonism for treating premature ejaculation.

Single exposure to social defeat increases corticotropin-releasing factor and glucocorticoid receptor mRNA expression in rat hippocampus.

Stressful life events are able to induce long-term modifications in physiological and neuroendocrine parameters that are related to the onset of several psychiatric disorders. To gain information on molecular modifications involved in long-term changes triggered by stress, we evaluated gene expression in the hippocampus of rats exposed to a single social defeat session. In the social defeat model, the experimental animal is defeated by a dominant male. The defeat induced an increase in body temperature, in distress vocalisations, in serum corticosterone levels and in anxiety-related behaviour measured with an open field test applied 6 h after the exposure to the dominant rat. In the open field test, anxiety-related behaviours were not detectable anymore 30 h after the exposure to the dominant rat and mRNA levels were evaluated at this time-point. The mRNA levels of genes modulated by stress (corticotropin-releasing factor; corticotropin-releasing factor receptor 1; corticotropin-releasing factor binding protein; mineralocorticoid and glucocorticoid receptors; Ca2+/calmodulin-dependent protein kinase-like kinase; Krox20; Bcl-2) and control genes (glyceraldehyde-3-phosphate dehydrogenase; beta-actin and cyclophilin A) were measured with real-time reverse transcription polymerase chain reaction. Corticotropin-releasing factor and glucocorticoid receptor mRNA levels were significantly modulated by the stress procedure, both genes showing an increase in rats exposed to a social defeat. No expression level differences were detected for the other genes. In conclusion, we report that 30 h after an acute social stress, a modification in mRNA levels can be detected in rat hippocampus, thus suggesting potential candidate genes involved in mediating long-term responses.

Intermittent exposure to social defeat and open-field test in rats: acute and long-term effects on ECG, body temperature and physical activity.

This study investigated the effects of exposure to an intermittent homotypic stressor on: (i) habituation of acute autonomic responsivity (i.e. cardiac sympathovagal balance and susceptibility to arrhythmias), and (ii) circadian rhythmicity of heart rate, body temperature, and physical activity. After implantation of a transmitter for the radiotelemetric recording of electrocardiogram (ECG), body temperature and physical activity, adult male rats (Rattus norvegicus, Wild Type Groningen strain) were repeatedly exposed (10 consecutive times, on alternate days) to either a social stressor (defeat by a con-specific, n = 15) or an open-field, control challenge (transfer to a new cage; n = 8). ECGs, body temperature and physical activity were continuously recorded in baseline, test and recovery periods (each lasting 15 min), at the 1st and 10th episodes of both defeat and open-field challenge. The circadian rhythms of heart rate, body temperature and physical activity were monitored before (5 days), during (16 days) and after (21 days) the intermittent stress protocol. This study indicates that there is no clear habituation of either acute cardiac autonomic responsivity (as estimated by means of time-domain indexes of heart rate variability) or arrhythmia occurrence to a brief, intermittent, homotypic challenge, regardless of the nature of the stressor (social or non-social). On the other hand, rats exposed to social challenge also failed to show adaptation of acute temperature and activity stress responsiveness, whereas rats facing open-field challenge developed habituation of activity and sensitization of temperature responses. Repeated social challenge produced remarkable reductions of the heart rate circadian rhythm amplitude (this effect being significantly greater than that produced by intermittent open-field), but only minor changes in the daily rhythms of body temperature and physical activity.